1. Technical Field
The present invention relates to a method of non-template-dependent and non-primer-dependent (hereinafter also referred to as "non-template and primer-dependent") synthesis of polydeoxyribonucleotides (DNA). The present invention further relates to a method of synthesizing chromosomal DNA and a method of producing chromosomes and, more particularly, it relates to a method of synthesizing chromosomal DNA and a method of producing chromosomes as established based on the findings that a certain deoxyribonucleotide polymerase (hereinafter briefly referred to as "DNA polymerase") can synthesize DNA not in the manner of "replication", i.e., to copy the genetic information of a template, but in the manner of "creation"; in other words, new pieces of genetic information are made. Some DNA synthesized in that manner serve as centromere, telomere and replication origin which are essential to chromosomal DNA.
2. Prior Art
So far, there have been two principal methods for the synthesis of DNA. One method uses DNA synthetase and the other is a chemical synthesis technique.
The method of synthesizing DNA using DNA synthetase includes so-called template-dependent methods of synthesizing DNA, such as the method of synthesizing DNA from template DNA using DNA polymerase and the method of synthesizing DNA from template RNAs using reverse transcriptase. However, these methods invariably require primers each comprising a short single-stranded DNA or RNA and a single-stranded template DNA for the synthesis of DNA. The so-called polymerase chain reaction (PCR) method, which is a method of amplifying template DNA in an easy and simple manner as developed recently Saiki et al., Science, volume 239, pages 487-491 (1988)!, still requires template DNA and primers.
The chemical method of DNA synthesis includes a phosphodiester method, a phosphotriester method and the like. However, these are complicated in synthetic process, require a lot of time and can synthesize only short DNA. Furthermore, the DNA synthesized are difficult to purify. While some apparatus are already commercially available for such synthesis, they have a drawback in that expensive and special reagents have to be used. The chemical method of synthesis, which has not yet been tried for the creation of DNA, would be substantially incapable of synthesizing DNA of a length of several thousand base pairs (bp). Even if such DNA could be successfully synthesized by said method, it would be expensive. Thus, it can of course be anticipated as well that the creation of proteins by means of the expression of the created DNA would be impossible.
The same applies to the synthesis of chromosomal DNA. Each chromosome possessed by a living organism is composed of a double-stranded linear DNA (chromosomal DNA) and proteins such as histones. Said double-stranded DNA has, as its essential elements, three regions for chromosomal functioning. Said three regions are (1) a centromere region (involved in appropriate partitioning of the replicated chromosome on the occasion of mitosis or meiosis), (2) a telomere region (involved in chromosome stabilization and replication) and (3) a replication start region (autonomously replicating sequence; ARS) (having the property of replication origin and which is involved in the initiation of DNA replication). Lack of any one of said regions makes said chromosomal DNA unstable and allows the disappearance of said DNA in the process of cell division. The total base sequences of the regions (1) to (3) mentioned above are more than 1,000 base pairs and, therefore, it is very difficult to synthesize them throughout to the end.
Therefore, in the prior art, the following technology of producing artificial chromosomes has been proposed. Thus, the technology proposed comprises excising a necessary portion alone from a DNA of living organism origin by a gene recombination technique and joining said portion to another DNA to produce an independent artificial chromosome. Joining, one by one, of the above-mentioned three regions essential to a chromosomal DNA would indeed make it possible to produce an artificial chromosome but the procedure leading thereto is very complicated and low in productivity.